Method for screening point mutation BIRC5 antigen epitope peptide

An antigen epitope and epitope peptide technology, applied in the field of molecular biology, can solve the problems of effective activation and expansion of T cells, low binding force, etc., achieve high accuracy, simple experimental process, and improve the effect of clinical trials

Pending Publication Date: 2021-11-05
GUANGDONG PHARMA UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Due to the relatively low binding force between self-antigens and MHC molecules under normal circumstances, it is difficult for TCR molecules on these low-af

Method used

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  • Method for screening point mutation BIRC5 antigen epitope peptide
  • Method for screening point mutation BIRC5 antigen epitope peptide
  • Method for screening point mutation BIRC5 antigen epitope peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0122] Bioinformatics analysis of embodiment 1 BIRC5 epitope

[0123] (1) NetMHCpan 4.1 online prediction system:

[0124] Log in to the website http: / / www.cbs.dtu.dk / services / NetMHCpan / , in "SUBMISSION", select "Fasta" for the "INPUT TYPE" item, enter the full amino acid sequence of BIRC5, and select "9mer" for the "PEPTIDE LENGTH" item peptides", select "HLA-A*02:01" in the "SELECT SPECIES / LOCI" project, and click "Submit" to run the remote prediction program.

[0125] The results were sorted according to the %Rank_EL score. The higher the %Rank_EL score, the weaker the binding ability of the epitope peptide to MHC molecules. We selected epitope peptides with 21<%Rank_EL score<30 for subsequent analysis. The list of epitope peptides with 21<%Rank_EL score<30 is shown in Table 1.

[0126] Table 1 NetMHCpan 4.1 prediction results of BIRC5 antigen 9 amino acid CTL epitope

[0127]

[0128] (2) SYFPEITHI online prediction system:

[0129] Login URL http: / / www.syfpeithi....

Embodiment 2

[0142] Example 2 Detection of the actual binding ability of the epitope peptide and the HLA-A2 molecule

[0143] Artificially synthesized candidate peptides include Sur79, Sur79I2 (mutant peptide), HIVpol 476 (negative peptide with high affinity with HLA-A2 molecules), Sur95 (positive peptide), a total of 4 groups.

[0144] (1) Detection of affinity between candidate epitope peptides and HLA-A2 molecules

[0145] Step Ⅰ) Take 1×10 for each group 6 / mL T2 cells were cultured overnight in DMEM medium containing 20% ​​FBS. After collecting the cells, they were washed twice with serum-free AIM-V medium and then resuspended and seeded in 24-well cell culture plates. Added 10 μM of different species The synthetic polypeptide and 3 μg / mL β2-MG were incubated at 37°C for 18 hours, and washed twice with PBS containing 3% FBS.

[0146] Step II) Resuspend the mixture obtained above in AIM-V medium for 2 hours at 37°C, collect cells, add FITC-labeled anti-HLA-A2 flow cytometry antibody...

Embodiment 3

[0157] Example 3 Establishment of CTLs clones induced by DC and mutant peptides in vitro

[0158] (1) DC differentiation induced in vitro

[0159] Firstly, prepare the special culture medium for induction and differentiation of DC in vitro: add 10% FBS, 150 μg / mL rhGM-CSF and 50 μg / mL rhIL-4 to RPMI-1640 liquid medium, mix well, and store at 4 °C for later use.

[0160] Collect the adherent mononuclear cells obtained before, and use DC-specific culture medium to divide the cells into 1×10 6 / mL density and seeded in 48-well plate, 0.5ml / well. The cells were cultured for 6 days at 37°C in an incubator with 5% CO2 saturated humidity. On the third day and the fifth day, half of the medium was changed. On the 8th day, 400 ng / mL rhTNF-α (10 ng / mL) was added to treat for 24 hours to stimulate DC maturation, and the obtained DCs were mature DCs (mDCs) for future use. The DC growth was observed with an inverted phase-contrast microscope, and the surface molecular markers of mature...

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Abstract

The invention relates to a method for screening point mutation BIRC5 antigen epitope peptides, and the method comprises the following specific steps: 1), comprehensively predicting CTL epitope peptides of BIRC5 antigen 9 amino acid residues by using NetMHCpan 4.1 and SYFPEITHI on-line prediction systems, and changing the types of corresponding amino acid residues through a point mutation technology to improve the score of on-line prediction; 2) according to the amino acid sequence obtained in the step 1), artificially synthesizing a corresponding epitope peptide, co-incubating the epitope peptide and T2 cells, and detecting the actual binding capacity of the epitope peptide and HLA-A2 molecules; 3) establishing specific CTLs cloning in vitro by utilizing the mutant peptide obtained by screening in the step (2), and identifying the immunological activity of the mutant peptide induced CTLs for specifically recognizing and killing target cells by utilizing ELISPOT experiment, calcein killing and other methods. The invention lays a foundation for further preparation of tumor peptide vaccines.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for screening point mutation BIRC5 antigen epitope peptides. Background technique [0002] The human BIRC5 gene is 14.7kb in full length, located on chromosome 17q25, and contains 4 exons and 3 introns. The gene is highly expressed in various tumor tissues, while its expression level is very low in normal differentiated tissues of the body BIRC5 It is a relatively recognized tumor antigen target with potential application value. [0003] Targeting tumor antigen targets, cytotoxic T lymphocytes are activated to exert anti-tumor effects by recognizing CTL epitope peptides bound to specific MHC-I molecules in specific tumor immunotherapy. Therefore, in many anti-tumor experimental studies, it is necessary to look for CD8 molecules that are stable in combination with MHC molecules and can effectively activate CD8. + The CTL epitope of T cells is very ...

Claims

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Application Information

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IPC IPC(8): G16B20/50G16B20/30G01N33/50G01N33/68C07K14/47
CPCG16B20/50G16B20/30C07K14/4748G01N33/68G01N33/5011
Inventor 沈晗董永坚叶婷刘静邵红伟
Owner GUANGDONG PHARMA UNIV
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